This proposal details the design, synthesis and application of a selective real-time Mitogen-Activated Protein Kinase (MAPK) activity probe. Phosphorylation plays a central role in the activation and deactivation of virtually all cellular processes. In particular, the MAPK signaling cascade produces a change in the observable phenotype of a cell by activating transcription factors in response to an extracellular stimulus. Recent work has shown that the use of CHelation-Enhanced Fluorescence can produce synthetic peptide-based phosphorylation sensors which undergo a 2- to 10-fold increase in fluorescence upon phosphorylation. These probes are modular in design and consist of an interchangeable kinase recognition motif, a phosphorylation site, and an unnatural amino acid. The modular nature of these probes allows for their extension to any kinase provided an appropriate recognition element is employed. Our specific aims are as follows: 1) Design, synthesis, and evaluation of a selective real-time MAPK activity sensor and 2) Delivery of the sensor into living cells. Thus this proposal will provide a fully modular synthetic set of kinase activity probes for use in real-time activity experiments in living cells. PUBLIC HEALTH RELEVANCE The Mitogen-Activated Protein Kinase (MAPK) pathway can stimulate a diverse set of cellular responses ranging from cell division to cell death;therefore even subtle changes in the regulation of this pathway can result in the acquisition of a disease state such as cancer. MAPKs are the terminal regulatory proteins in this pathway and as such are of great interest to the scientific and medical communities. The development of a selective real-time MAPK activity probe would aid in the evaluation of the duration and intensity of MAPK activity in response to external factors.